1. Field of the Invention
The present invention relates to a microfluidic separation device, a separation method using the same and a kit for separating circulating rare cells from blood using the same, and more particularly, to a microfluidic-based separation technology for fixing target particles of a sample, which have a specific affinity for magnetic nanoparticles, to a device by use of a magnetic material, and for isolating the sample from which the target particles have been removed.
2. Description of Related Art
The present invention can be effectively applied to remove leukocytes from a blood sample in order to isolate circulating rare cells (CRCs), particularly circulating tumor cells (CTCs).
In general, a biochemical sample exists as a mixture of two or more components, and thus separation technology for either analyzing only a desired component in the mixture or purifying only a specific component from the mixture is very important in a sample pretreatment process. In particular, lab-on-a-chip technology for processing a small amount of a sample at high speed and with high efficiency by use of the microfluidic channel, mixer, pump, valve and the like integrated on a single chip is receiving attention.
Furthermore, cell-based diagnostics, which is important in biological or medical analysis, involves blood analysis, cell research, microbial analysis and the like. With the recent development of cell research and analysis techniques and protein and DNA analysis techniques, studies have been conducted on unify and integrate such clinical diagnostic procedures in the form of a microfluidic device.
Microfluidic technology is a technology for handling a small amount (10−6-10−12 liter) of a sample. It has high sensitivity despite a small sample amount, and can be easily combined with other technologies to maximize its efficiency. In addition, it is easily constructed and is inexpensive. Due to such advantages, the microfluidic technology is used in various fields.
Circulating rare cells (CRCs) are cells circulating in cells, and are very rarely present at a concentration of less than 1000 cells per ml of blood. Such circulating rare cells include circulating tumor cells (CTCs), nucleated red blood cells (nRBCs), circulating endothelial cells (CECs) and circulating stem cells (CSCs), which can be used as indicators for the early diagnosis and prognostic diagnosis of various diseases.
Particularly, in recent years, there have been increasing studies on detecting blood tumor cells, which are epithelial cells isolated from tumor, to early diagnose metastatic cancer or to monitor the results of cancer therapy. This method has the advantage of not requiring biopsy such as isolating tumor tissue directly from the body, and thus is absolutely advantageous for lung cancer patients in which tissue biopsy is difficult. However, because circulating tumor cells (CTCs) in the blood of patients are present at a very low concentration (about 1 CTC/109 blood cells), there is much difficulty in efficiently capturing and detecting the circulating tumor cells.
Korean Patent Laid-Open Publication No. 10-2013-0107583 (entitled “Composition for diagnosis of tumor cells in blood and method for detecting tumor cells in blood using the same”; published on Oct. 2, 2013) discloses a composition for diagnosis of tumor cells in blood, the composition comprising: nanoparticles having attached thereto a primary antibody such as an EpCAM antibody, which binds specifically to blood tumor cells in order to the efficiency of capture of blood tumor cells and minimize non-specific binding to blood cells to thereby increase the limit of detection; magnetic beads having a size ranging from 100 nm to 1 μm and having attached thereto a secondary antibody such as protein A, which binds to the primary antibody.
Meanwhile, a conventional magnetic-activated cell sorting (MACS) method based on magnetic nanoparticles (MNPs) is performed by mixing a magnetic particle-containing solution with a particle-containing solution using a pipette, allowing the mixture to stand for a certain time to form an MNP complex, transferring the MNP complex into a container surrounded by a magnet, and then gently sucking particles, which have not attracted to the wall surface of the container, with a pipette after a certain time. However, this conventional method has a problem in that, because it comprises multiple stages which are all performed by a manual process, particles to be sorted can be lost.